Compositions and Methods for Treatment of Prostate and Other Cancers

ABSTRACT

Therapeutic agents which target heat shock protein (hsp) 27 in vivo are used to provide treatment to individuals, particularly human individuals, suffering from prostate cancer and other cancers that overexpress hsp27. A therapeutic agent, for example an antisense oligonucleotide or RNAi nucleotide inhibitor with sequence specificity for hsp27 mRNA, for example human hsp27 mRNA, is administered to an individual suffering from prostate cancer or some other cancer expressing elevated levels of hsp27 in a therapeutically effective amount. The therapeutic agent is suitably formulated into a pharmaceutical composition which includes a pharmaceutically acceptable carrier, and packaged in dosage unit form. A preferred dosage unit form is an injectable dosage unit form.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional ApplicationsNos. 60/415,859 filed Oct. 2, 2002 and 60/463,952 filed Apr. 18, 2003,both of which are incorporated herein by reference.

BACKGROUND OF INVENTION

This application relates to compositions and methods for the treatmentof prostate and other cancers that express elevated levels of hsp 27 ascompared to normal tissue in at least some stages of diseasedevelopment.

Prostate cancer is the most common cancer that affects men, and thesecond leading cause of cancer deaths in men in the Western world.Because prostate cancer is an androgen-sensitive tumor, androgenwithdrawal, for example via castration, is utilized in some therapeuticregimens for patients with advanced prostate cancer. Androgen withdrawalleads to extensive apoptosis in the prostate tumor, and hence to aregression of the disease. However, castration-induced apoptosis is notcomplete, and a progression of surviving tumor cells toandrogen-independence ultimately occurs. This progression is the mainobstacle to improving survival and quality of life, and efforts havetherefore been made to target androgen-independent cells. These effortshave focused on non-hormonal therapies targeted againstandrogen-independent tumor cells (Yagoda et al., Cancer 71 (Supp. 3):1098-1109 (1993); Oh et al., J. Urol 60: 1220-1229 (1998)), however, sofar no non-hormonal agent has improved survival. Alternative approachesare therefore indicated.

It has been observed that numerous proteins are expressed in increasedamounts by prostate tumor cells following androgen withdrawal. At leastsome of these proteins are assumed to be associated with the observedapoptotic cell death which is observed upon androgen withdrawal. (Raffoet al., Cancer Res.: 4448-4445 (1995): Krajewska et al., Am. J. Pathol.148: 1567-1576 (1996); McDonnell et al., Cancer Res. 52: 6940-6944(1992)).

SUMMARY OF INVENTION

The present invention makes use of therapeutic agents which target heatshock protein (hsp) 27 in vivo to provide treatment to individuals,particularly human individuals, suffering from prostate cancer and othercancers that overexpress hsp27. In accordance with the invention, atherapeutic agent, for example an antisense oligonucleotide or RNAinucleotide inhibitor with sequence specificity for hsp27 mRNA, forexample human hsp27 mRNA, is administered to an individual sufferingfrom prostate cancer or some other cancer expressing elevated levels ofhsp27 in a therapeutically effective amount. The therapeutic agent issuitably formulated into a pharmaceutical composition which includes apharmaceutically acceptable carrier, and packaged in dosage unit form. Apreferred dosage unit form is an injectable dosage unit form.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-G show results of mRNA expression tests in cells exposed toantisense oligonucleotides of Seq. ID Nos. 1-81.

FIG. 2 shows the effect of hsp27 antisiense on hsp27 expression in PC3cells.

FIGS. 3A and 3B show tumoral volume and serum PSA after treatment withhsp27 anstisense.

FIGS. 4A and 4B show changes in tumor volume following treatment withhsp27 antisense with and without taxol.

FIG. 5 shows reduction of hsp27 mRNA following treating with RNAi.

FIGS. 6A and 6B show the amount of expressed hsp27 protein followingtreatment with RNAi.

FIGS. 7A-7C shows results of antisense and RNAi treatment of prostatecancer cells.

FIG. 8 shows hsp27 expression in T24 bladder cancer cells.

FIG. 9 shows immunoreactivity of hsp27 determined fromimmunohistological evaluation of hsp27 in an NHT tissue array.

DETAILED DESCRIPTION

The present invention relates to compositions that reduce the effectiveamount of active hsp27 in vivo. Exemplary compositions useful in theinvention are antisense hsp27 oligonucleotides or RNAi nucleotideinhibitors. The invention further relates to the use of thesecompositions in the treatment of prostate cancer and other cancers thatexpress hsp27 in elevated amounts.

As used in the specification and claims of this application, the term“active hsp27” refers to hsp27 which is active as a chaperone tostabilize protein structure at times of stress and in particularinhibits the activity of caspase-3, a mediator of apoptosis. Reductionin levels of active hsp27 can be achieved by reducing the total amountof hsp27, either by restricting production of hsp27 or by degradinghsp27 at a rate faster than it is being produced, by converting hsp27 toan inactive form, for example by sequestering hsp27 in an inactivecomplex such as with an anti-hsp27 antibody.

As used in the specification and claims hereof, the cancers which may betreated are those that express hsp27 in an elevated amounts compared tonon-cancerous cells of the same tissue type. Exemplary cancers includewithout limitation prostate, bladder, lung, breast, osteosarcoma,pancreatic, colon, melanoma, testicular, colorectal, urothelial, renalcell, hepatocellular, leukemia, lymphoma, and ovarian cancer and centralnervous system malignancies.

As used in the specification and claims hereof, the term “sequencespecificity” refers to the existence of a complementary relationship,using Watson-Crick base pairing, between the oligonucleotide and thehsp27 target that is sufficient to yield specific binding underintra-cellular conditions. Perfect complementarity is desirable, but isnot absolutely required, particularly where longer oligonucleotides areemployed.

The sequence of human hsp27 mRNA is known, for example from NCBIAccession Numbers AB020027, X54079, NM_(—)006308, NM_(—)001540 andNM_(—)001541. The cDNA sequence (Seq. ID No. 91) forms the basis for thedevelopment of antisense oligonucleotides and RNAi nucleotideinhibitors. The preferred sequences for antisense, and for RNAi arethose that target bases in the regions from nucleotides 131-161,241-261, 361-371, 551-580, 661-681 and 744-764 in Seq. ID No. 91. INorder to target bases within these regions, an antisense or RNAimolecule must have sequence specificity with a region that includes atleast one of the listed bases, preferably at least 10 of the listedbases.

Suitable antisense oligonucleotides have a length of from 12 to 35oligonucleotides and have sequence specificity to the hsp27 mRNAsequence. Antisense oligonucleotides that were made and tested for theirability to reduce the amount of active hsp27 mRNA are set forth as SeqID Nos. 1 to 82. Preferred antisense oligonucleotides have the sequence5′-ggggacgcggcgctcggtcat-3′ (Seq. ID No. 81) or5′-gggacgcggcgctcggtcat-3′ (Seq. ID No. 82) which targets thetranslation initiation site of hsp27 mRNA, as well as those with Seq. IDNos. 25, 36, 56, 57, 67 and 76.

RNA interference or “RNAi” is a term initially coined by Fire andco-workers to describe the observation that double-stranded RNA (dsRNA)can block gene expression when it is introduced into worms (Fire et al.(1998) Nature 391, 806-811, incorporated herein by reference). dsRNAdirects gene-specific, post-transcriptional silencing in many organisms,including vertebrates, and has provided a new tool for studying genefunction. RNAi involves mRNA degradation, but many of the biochemicalmechanisms underlying this interference are unknown. The use of RNAi hasbeen further described in Carthew et al. (2001) Current Opinions in CellBiology 13, 244-248, and Elbashir et al. (2001) Nature 411, 494-498,both of which are incorporated herein by reference. The RNAi moleculesof the invention are double-stranded or single-stranded RNA of fromabout 21 to about 23 nucleotides which mediate RNA inhibition. That is,the isolated RNAi of the present invention mediate degradation of mRNAof the hsp27 gene.

The terms RNA, RNA molecule(s), RNA segment(s) and RNA fragment(s) maybe used interchangeably to refer to RNA that mediates RNA interference.These terms include double-stranded RNA, single-stranded RNA, isolatedRNA (partially purified RNA, essentially pure RNA, synthetic RNA,recombinantly produced RNA), as well as altered RNA that differs fromnaturally occurring RNA by the addition, deletion, substitution and/oralteration of one or more nucleotides. Such alterations can includeaddition of non-nucleotide material, such as to the end(s) of the RNA orinternally (at one or more nucleotides of the RNA). Nucleotides in theRNA molecules of the present invention can also comprise non-standardnucleotides, including non-naturally occurring nucleotides ordeoxyribonucleotides. Collectively, all such altered RNAi compounds arereferred to as analogs or analogs of naturally-occurring RNA. RNA of thepresent invention need only be sufficiently similar to natural RNA thatit has the ability to mediate RNAi. As used herein the phrase “mediateRNAi” refers to and indicates the ability to distinguish which mRNA areto be affected by the RNAi machinery or process. RNA that mediates RNAiinteracts with the RNAi machinery such that it directs the machinery todegrade particular mRNAs or to otherwise reduce the expression of thetarget protein. In one embodiment, the present invention relates to RNAmolecules that direct cleavage of specific mRNA to which their sequencecorresponds. It is not necessary that there be perfect correspondence ofthe sequences, but the correspondence must be sufficient to enable theRNA to direct RNAi inhibition by cleavage or lack of expression of thetarget mRNA.

As noted above, the RNA molecules of the present invention in generalcomprise an RNA portion and some additional portion, for example adeoxyribonucleotide portion. The total number of nucleotides in the RNAmolecule is suitably less than 49 in order to be effective mediators ofRNAi. In preferred RNA molecules, the number of nucleotides is 16 to 29,more preferably 18 to 23, and most preferably 21-23.

The RNA portion of suitable RNAi molecules are set forth in Seq. ID Nos.83-90. These sequences are the sense RNA strand. They may be used inRNAi treatment in combination with a corresponding antisense strand.

The oligonucleotides employed as antisense or RNAi molecules may bemodified to increase the stability of the oligonucleotides in vivo. Forexample, the oligonucleotides may be employed as phosphorothioatederivatives (replacement of a non-bridging phosphoryl oxygen atoms witha sulfur atom) which have increased resistance to nuclease digestion.MOE modification (ISIS backbone) is also effective.

Administration of antisense oligonucleotides can be carried out usingthe various mechanisms known in the art, including naked administrationand administration in pharmaceutically acceptable lipid carriers. Forexample, lipid carriers for antisense delivery are disclosed in U.S.Pat. Nos. 5,855,911 and 5,417,978 which are incorporated herein byreference. In general, the antisense is administered by intravenous,intraperitoneal, subcutaneous or oral routes, or direct local tumorinjection.

The amount of antisense oligonucleotide or other therapeuticadministered is one effective to reduce the amount of active hsp27. Itwill be appreciated that this amount will vary both with theeffectiveness of the antisense oligonucleotides or other therapeuticagent employed, and with the nature of any carrier used. Thedetermination of appropriate amounts for any given composition is withinthe skill in the art, through standard series of tests designed toassess appropriate therapeutic levels.

The RNAi molecules of the invention are used in therapy to treatpatients, including human patients, that have cancers or other diseasesof a type where a therapeutic benefit is obtained by the inhibition ofexpression of the targeted protein. siRNA molecules of the invention areadministered to patients orally, by one or more daily injections(intravenous, subcutaneous, intravesical, or intrathecal) or bycontinuous intravenous or intrathecal administration for one or moretreatment cycles to reach plasma and tissue concentrations suitable forthe regulation of the targeted mRNA and protein.

Prostate cancer is one cancer that overexpresses hsp27 in later stagecancers, and in particular in cancers that have become androgenindependent. FIG. 9 shows immunoreactivity of hsp27 determined fromimmunohistological evaluation of hsp27 in an NHT tissue array. In thebenign samples, immunoreactivity is limited to the basal layer. As theduration of neoadjuvant therapy increases, the immunoreactivityincreases, with androgen indepenent tumors showing very strongreactivity. For treatment of prostate cancer, the therapeuticcompositions of the invention are suitably administered after initial ofandrogen withdrawal. Initiation of androgen withdrawal may beaccomplished via surgical (removal of both testicles) or medical(drug-induced suppression of testosterone) castration, which iscurrently indicated for treatment of prostate cancer. Medical castrationcan be achieved by various regimens, including LHRH agents orantiandrogens. (Cleave et al., CMAJ160: 225-232 (1999)). Intermittenttherapy in which reversible androgen withdrawal is effected is describedin Cleave et al. Eur. Urol. 34 (Supp. 3): 37-41 (1998).

The inhibition of hsp 27 expression may be transient, and for treatmentof prostate cancer ideally should occur coincident with androgenwithdrawal. In humans, this means that inhibition of expression shouldbe effective starting within a day or two of androgen withdrawal (beforeor after) and extending for about 3 to 6 months. This may requiremultiple doses to accomplish. It will be appreciated, however, that theperiod of time may be more prolonged, starting before castration andexpending for substantial time afterwards without departing from thescope of the invention.

The method for treating cancer, including prostate cancer, in accordancewith the invention may further include administration of chemotherapyagents and/or additional antisense oligonucleotides directed atdifferent targets. Examples of other therapy agents include, withoutlimitation, taxanes (paclitaxel or docetaxel), mitoxanthrone, andantisense directed to Bcl-2, Bcl-xl or c-myc. Inhibition of hsp27 usingantisense or RNAi can be used to enhance the activity of like taxanes orgemcitabine, as well as biologic agents for the treatment of prostate,breast, lung, urothelial and other cancers.

The invention will now be further described with respect to thefollowing non-limiting examples.

Example 1

A plurality of antisense compounds as defined in Seq. ID Nos. 1-81 wereprepared, and each sequence was tested for levels of Hsp 27 mRNAexpression human prostate cancer PC3 cells by Northern Blot afterexposure to 50 nM of a specified antisense oligonucleotide in anOligofectamine carrier. The results of these tests, as a percentage ofan Oligofectamine only control, for Seq. ID Nos. 1-81 are shown in FIGS.1A-G. As shown, although not all antisense sequences are effective,effective antisense sequences are found throughout the length of thehsp27 mRNA.

Example 2

PC3 prostate cancer cells were transfected at 40% confluency with threeconcentrations (10, 30 and 50 nM) of 6 different hsp27-antisenseoligonucleotides 2 times, successively in 10 cm dishes, using anOligofectamine carrier. RNA was extracted 48 hours after the firsttreatment and analyzed by Northern Blot. The antisense oligonucleotidestested were those with Seq. ID Nos. 67, 57, 25, 76, 56 and 36. Ascontrols, a scrambled oligonucleotide and Oligofectamine onlyexperiments were conducted. All of the oligonucleotides tested showeddown-regulation of hsp27 with respect to the controls at least at one ofthe concentrations. Seqs. ID 71 and 74 appeared to be most effective,with significant down-regulation at 10 nM. The results, relative to aGAPDH control are depicted graphically in FIG. 2.

Example 3

Xenografts of LNCaP prostate cancer cells were introduced into mice, andthe effect of intraperitoneal injection of hsp27-antisenseoligonucleotide (Seq. ID No. 82) administered intraperitoneally, 10mg/kg., once daily for four weeks following androgen withdrawal bycastration was evaluated. As shown in FIGS. 3A and 3B, tumoral volumeand serum PSA increased in the weeks following treatment with ascrambled control, indicating progression to androgen independence, andthus, the loss of efficacy of the castration therapy. In contrast, thisprogression to androgen independence was not observed in the same timeperiod when treatment with the hsp27 antisense oligonucleotide wasgiven.

Example 4

Xenografts of PC3 prostate cancer cells were introduced into mice, andthe effect of intraperitoneal injection of hsp27-antisenseoligonucleotide (Seq. ID No. 82) administered intraperitoneally, 10mg/kg., once daily for four weeks with and without Taxol was evaluated.As shown in FIGS. 4A and 4B, tumor volume was significantly reduced bytreatment with hsp27-antisense, as compared to scrambledoligonucleotide. This effect was enhanced when taxol treatment wascombined with the antisense treatment. FIG. 4A illustrates single agentanti-tumor activity while FIG. 4B illustrates that administration ofhsp27 antisense can sensitize cells to paclitaxel in vivo. The controlin 4B is scrambled plus taxol.

Example 5

RNAi molecules having a sequence in accordance with Seq. ID Nos. 84, 85,87, 88 and 90 were tested in PC3 cells. The PC cells were transfectedwith various amounts of the hsp27 siRNA or scrambled control. Two daysafter transfection, total RNA was extracted and analyzed by Northernblotting for hsp27 and 285 levels. Cells treated with Oligofectimineonly were used as an additional control. FIG. 5 shows densitometricmeasurements of hsp27 mRNA after normalization to 285 mRNA controls. Asshown, Seq. ID Nos. 84, 85, 87, 88 and 90 are all effective tosignificantly reduce hsp27 expression as compared to the scrambledcontrol.

Example 6

RNAi having a sequence in accordance with Seq ID. No. 84 was transfectedinto PC3 cells, and the amount of expressed hsp27 protein, as a comparedto Vinculin expression was determined. The results are shown in FIGS. 6Aand 6B. As shown, a dose dependent reduction in hsp27 expression isobserved following treatment with the RNAi molecule.

Example 7

LNCaP cells (10⁴ cells/well, cultured in 12-well plates) weretransfected in vitro with 1 nM RNAi having a sequence in accordance withSeq ID. No. 84. Cell growth was monitored using a Crustal Violet assay.As shown in FIG. 7A, the RNAi treatment resulted in a reduction in cellgrowth as compared to treatment with Oligofectamine only or a scrambledcontrol. The experiment was repeated using PC3 cells. FIG. 7B shows the% of cells alive 3 days after transfection. FIG. 7C shows growthinhibition of PC3 cells in vitro after treatment with hsp27 antisenseSeq. ID NO. 82

Example 8

Human bladder cancer T24 cells transfected with hsp27 antisense (Seq. IDNo 82) or RNAi (Seq. ID No. 84) were tested for hsp27 expression. Asshown in FIG. 8, RNAi and the antisense were both effective to reducethe amount of hsp27 expressed in these cells.

1-24. (canceled)
 25. A pharmaceutical composition comprising atherapeutic agent effective to reduce the amount of active hsp27 incancerous cells exposed to the therapeutic agent, and a pharmaceuticallyacceptable carrier, wherein the therapeutic agent is an antisenseoligonucleotide having a sequence complementary to SEQ. ID NO. 91,wherein the oligonucleotide comprises at least ten bases complementaryto bases 744-764 of SEQ. ID NO. 91, and wherein the antisenseoligonucleotide is 12 to 35 nucleotides in length.
 26. The compositionof claim 25, wherein the composition is packaged in dosage unit form.27. The composition of claim 26, wherein the dosage unit form is aninjectable solution.
 28. The composition of claim 25, wherein theantisense oligonucleotide is modified to increase in vivo stability. 29.The composition of claim 25, wherein the therapeutic agent is anantisense oligonucleotide that comprises the sequence of bases as setforth in SEQ. ID NO.
 76. 30. The composition of claim 29, wherein thecomposition is packaged in dosage unit form.
 31. The composition ofclaim 30, wherein the dosage unit form is an injectable solution. 32.The composition of claim 29, wherein the therapeutic agent is anantisense oligonucleotide that comprises the sequence of bases as setforth in SEQ. ID NO.
 76. 33. A pharmaceutical composition comprising a(a) means for reducing the amount of active hsp27 in cancerous cells bysequence specific interaction with Seq. ID No. 91 and (b) apharmaceutically acceptable carrier.
 34. The pharmaceutical compositionof claim 33, wherein the means for reducing the amount of active hsp27in cancer cells is an oligonucleotide, and the oligonucleotide consistsof 12 to 35 nucleotides.
 35. The pharmaceutical composition of claim 34,wherein the oligonucleotide is an antisense oligonucleotidecomplementary to Seq. ID No. 91.